Adaptor-Specific Antibody Fragment Inhibitors for the Intracellular Modulation of p97 (VCP) Protein–Protein Interactions

Protein–protein interactions (PPIs) form complex networks to drive cellular signaling and cellular functions. Precise modulation of a target PPI helps explain the role of the PPI in cellular events and possesses therapeutic potential. For example, valosin-containing protein (VCP/p97) is a hub protein that interacts with more than 30 adaptor proteins involved in various cellular functions. However, the role of each p97 PPI during the relevant cellular event is underexplored. The development of small-molecule PPI modulators remains challenging due to a lack of grooves and pockets in the relatively large PPI interface and the fact that a common binding groove in p97 binds to multiple adaptors. Here, we report an antibody fragment-based modulator for the PPI between p97 and its adaptor protein NSFL1C (p47). We engineered these antibody modulators by phage display against the p97-interacting domain of p47 and minimizing binding to other p97 adaptors. The selected antibody fragment modulators specifically disrupt the intracellular p97/p47 interaction. The potential of this antibody platform to develop PPI inhibitors in therapeutic applications was demonstrated through the inhibition of Golgi reassembly, which requires the p97/p47 interaction. This study presents a unique approach to modulate specific intracellular PPIs using engineered antibody fragments, demonstrating a method to dissect the function of a PPI within a convoluted PPI network.

. Selection process with Fab-phage libraries. Figure S2. Biolayer interferometry (BLI) dose response profiles of scFv binders to p47-UBX domain. Figure S3. BLI results of selected scFv binders to the interacting domains of p97 adaptor proteins. Figure S4. Amino acid sequence alignment between p37-UBX and p47-UBX and their key binding residues with p97-N domain. Figure S5. BLI dose response profiles of scFv-A06 and scFv-E04 to p37-UBX domain.         S8 Figure S6. Surface plasmon resonance (SPR) sensorgrams for the scFv-binder/p47-UBX mixture binding to full-length human p97 in the presence of either 100 μM ATP or 100 μM ADP. In the mixture, the concentration of p47-UBX was fixed at 50 nM with an increasing concentration of scFv binders. Data represent N = 2 independent experiments. S9 Figure S7. (a) Schematic illustration of p47 constructs in this study. (b~i) Surface plasmon resonance (SPR) sensorgrams for the scFv-binder/p47-UBX or the scFv-binder/p47 mixture binding to full-length human p97 in the presence of either 100 μM ATP or 100 μM ADP. In the mixture, the concentration of p47-UBX or p47 was fixed at 50 nM with an increasing concentration of scFv binders.  Western blots for HA-tag, p47, p97, and vinculin in U2OS cells after 24-h transfection of plasmids that encode (b) scFvs and (c) scFabs. For each group, input proteins in the lysates (IN) before the co-IP of p47 were compared with the unbound proteins in the lysates (UNB). Ctrl, cell-only control group without Xfect-plasmid complexes added. Data represent N = 2 independent experiments. S11 Figure S10. (a) Co-IP of p97 from U2OS cells and (b) western blots for p97, p47, HA-tag, and vinculin in U2OS cells after 24-h transfection of plasmids that encode scFvs. The interactions between p97 and its more than 30 adaptor proteins are exceptionally dynamic, SR2 resulting in unsuccessful capture of most p97/adaptor complexes in the co-IP experiments of p97.
Moreover, the formation of a trimeric complex was observed among p97, p47, and scFv-A06 upon the introduction of antibody fragment inhibitors in U2OS cells. For each group of western blots, input proteins in the lysates (IN) before the co-IP of p97 were compared with the unbound proteins in the lysates (UNB). Ctrl, cell-only control group without Xfectplasmid complexes added.  have been tagged with NanoLuc (NLF1) donor or HaloTag (HT) acceptor at either N-or C-terminus. All the combinations were transfected in a ratio of wplasmid : wplasmid = 1:1. (b) NanoBRET assay results in wild-type U2OS cells testing varied ratios between pHTC-p47 and pNLF1N-p97. Note that all the p97 and p47 constructs in (a) and (b) were tagged with a nuclear export signal (NES). (c) NanoBRET assay results in p47-KO U2OS cells demonstrate that overexpressed p47-FLAG suppresses the BRET ratio when compared to empty vectors (EV), indicating the competition between p47-FLAG and pHTC-p47 for pNLF1N-p97 binding. (d) NanoBRET assay results in p47-KO U2OS cells testing varied ratios between pHTC-p47-UBX and pNLF1N-p97-N. N = 4 for all NanoBRET assays. S14 Figure S13. Representative immunofluorescence images of a Golgi marker GRASP55 in HeLa cells transfected with anti-p47-UBX antibody fragments (HA-tagged). Scale bar, 10 µm. Figure S14. (a) Co-IP of p47 from HeLa cells and (b) western blots for HA-tag, p47, p97, and vinculin in HeLa cells after 24-h transfection of plasmids that encode scFab-NLS-A06, scFab-NLS-E04, scFab-NLS-A06/E04 (wplasmid / wplasmid = 1:1), and EGFP-NLS. (c) Co-IP of p47 from Rat-1 or NRK cells and (d) western blots for HA-tag, p47, p97, and vinculin in Rat-1 or NRK cells after 24-h transfection of plasmids that encode scFab-NLS-A06 and scFab-NLS-E04. For each group of western blots, input proteins in the lysates (IN) before the co-IP of p47 were compared with the unbound proteins in the lysates (UNB). Ctrl/Control, cell-only control group without Xfect-plasmid complexes added. Data represent N = 2 independent experiments. S16

Plasmid construction
The plasmid constructs in this work were obtained by PCR amplifications of DNA fragments, followed by Gibson assemblies of the DNA fragments. P97, p47, p47-UBX, NPL4-UBXL, FAF1-UBX, and p37-UBX were cloned on E. coli expression vectors (pET) and expressed as biotinylated proteins as previously described.  These constructs for biotinylation contain a TEV (tobacco etch virus) protease cleavage site, an AviTag, and a His-tag (6 aa, HHHHHH) at either the N-terminus or the C-terminus. All the scFvs were subcloned from the Fab-phagemid into an E. coli expression vector pSYN1 SR5 and a mammalian expression vector pcDNA3.1. All scFabs SR6 were designed from the scFv constructs by overlap-extension PCR SR7 and subcloned into a pcDNA3.1 vector. The scFab-NLS constructs were subcloned from their corresponding scFab constructs with an N-terminal c-Myc NLS tag SR8 (9 aa, PAAKRVKLD). All the constructs on pcDNA3.1 vectors in this study contain a C-terminal HA (human influenza hemagglutinin) epitope tag (9 aa, YPYDVPDYA). All constructs were sequence verified by Sanger sequencing.

Protein expression and purification
All the recombinant proteins were expressed in E. coli BL21(DE3) and purified as previously described.  Briefly, E. coli containing expression vectors of interest were grown in 2X YT media at 37 °C to an OD600 of 0.4~0.8, followed by the addition of 0.1 mM IPTG at 20 °C overnight. Bacterial cells were pelleted by centrifugation and lysed with either sonication probe or B-PER lysis buffer. Next, His-tagged proteins were purified on a Ni-NTA column, followed by size-exclusion chromatography (SEC) using a GE AKTA FPLC purification system equipped with a Superdex column. When the biotinylated tag was not needed for specific experiments, the biotinylated proteins were cleaved by TEV protease (NEB, Cat# P8112S), followed by dialysis and SEC purification. The purified fractions were identified by SDS-PAGE and characterized by mass spectrometry on a Waters Xevo QTof mass spectrometer. Fractions containing the protein of interest were concentrated by Amicon MWCO filters, flash frozen, and stored at -80 °C.

Fab-phage selection
Phage display was carried out based on previously established protocols. SR9 Briefly, the antibody phage Library E or X7X8 were used for selection against biotinylated p47-UBX domain (antigen). After incubating the phage library with the antigen, streptavidin-functionalized magnetic beads were utilized to capture the bound phage, followed by the elution step using TEV protease. Four rounds of selections were conducted with a decreasing concentration of the antigen (500 nM, 250 nM, 100 nM, and 10 nM). Note that during the third and fourth rounds, the overnight phage culture was pre-enriched using Protein-L magnetic beads (ThermoFisher, Cat# 88849) before the antigen capture. The clones from the fourth round of selection were collected for phage ELISA analysis.

Phage ELISA
Phage ELISA was performed based on previous reports. SR9 For each phage clone, three different conditions were tested -Direct: biotinylated p47-UBX; Competition: biotinylated p47-UBX with an equal concentration of p47-UBX (w/o biotinylation) in solution; Control: BSA (bovine serum albumin). A 384-well Nunc Maxisorp flat-bottom clear plate (Thermo Scientific, Cat# 464718) was coated with 10 μg·mL -1 of NeutrAvidin (Thermo Scientific, Cat# 31000) in PBS overnight at 4 °C and subsequently blocked with PBSTB (PBS buffer + 0.02% Tween-20 + 0.2% BSA) for 1 hour at room temperature. Plates were washed three times with PBS containing 0.05% Tween-20 and were washed similarly between each step. Next, 20 nM of biotinylated p47-UBX diluted in PBSTB was captured on the NeutrAvidin-coated wells (for Direct and Competition wells; PBSTB for the Control wells) for 30 min, then blocked with PBSTB + 20 μM biotin for 10 min. After washing the plate, phage supernatant diluted (5 times) in PBSTB was added for 1 hour at 4 °C for the Direct and Control groups. For the Competition groups, the phage supernatant was diluted into PBSTB with 20 nM soluble p47-UBX and incubated for 1 hour at 4 °C. Bound phage was detected by incubating with anti-M13-horseradish peroxidase conjugate (Sino Biologics, Cat#11973-MM05-H, 1:5000) for 30 min, followed by the addition of TMB substrate (VWR, Cat# 95059-156) and incubation at room temperature until signal appeared. The reaction was quenched by the addition of 1 M S18 phosphoric acid. The absorbance at 450 nm was measured using a SpectraMax plate reader. The ELISA data were analyzed to refine hits with the two following features: (1) Specificity = OD450 nm (Direct)/OD450 nm (Control) > 4; (2) Competition ratio = OD450 nm (Competition)/OD450 nm (Direct) < 0.5.
Data were analyzed using the ForteBio Octet RED384 Data Analysis HT software and binding affinities (KD) were determined using a 1:1 monovalent binding model.
Note that all the primary and secondary antibodies for IF experiments were diluted in PSBT that contains 1% BSA. After washing with PBS, the cells were imaged in PBS on a Nikon Ti confocal microscope equipped with a Yokagawa CSU22 S20 spinning disk unit. The intracellular distribution was measured with excitation wavelengths of 488 nm (Alexa Fluor 488) and 561 nm (Alexa Fluor 647).

Co-immunoprecipitation (co-IP) and western blot
Transfection and lysate preparation. Mammalian cells (a total of 120 k for U2OS and HeLa cells, 150 k for Rat-1 and NRK cells) in complete DMEM media (w/o antibiotics) were cultured in a 6-well plate (Corning,Cat# 3516) for 24 hours prior to the experiment. The complexes between 1.5 μL Xfect transfection reagent (Takara Bio, Cat# 631318) and 5 μg plasmids (for the expression of antibody fragments) were mixed in 100 μL Xfect reaction buffer for 10 min. After replacing the media with 900 μL fresh complete DMEM media (w/o antibiotics), the Xfect-plasmid mixture was added dropwise into the well and incubated at 37 °C for 24 hours. Next, the complex-containing media was aspirated and the cells were washed once with cold PBS. After aspirating the PBS, 300 μL of NP-40 lysis buffer [50 mM Tris·HCl pH 7.5, 150 mM NaCl, 0.5% NP-40 substitute (Sigma-Aldrich, Cat# 74385), 1 tablet of protease inhibitor (Sigma-Aldrich, Cat# 05892791001) per 10-mL buffer] was added to the cells and the plate was placed on an orbital shaker at 4 °C for 5 min. Each well was then thoroughly scraped with a 1-mL pipette tip. The lysate suspension in each well was collected and centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was collected as the cell lysates for co-IP and western blot analysis.
Co-IP and western blot. The cell lysates were mixed with 3 μL rabbit anti-p47 antibodies (ThermoFisher, Cat# PA5-61429) and incubated overnight at 4 °C. Meanwhile, 50 μL Protein-A magnetic beads (ThermoFisher, Cat# 88845) were prewashed with NP-40 lysis buffer. Next, 200 μL of the lysates were incubated with the pre-washed beads at room temperature on an orbitron rotator, and the remaining lysates were labeled as the INPUT group. After an hour of incubation, the beads were collected by a magnet (ThermoFisher, Cat# 12321D) and the lysates containing the unbound proteins were collected (the UNBOUND group). The beads with p47-bound complexes were heated at 90 °C for 5 min in 1X LDS sample buffer (ThermoFisher, Cat# B0007; diluted in NP-40 cell lysis buffer), resulting in the elution as the co-IP portion of the corresponding group. For the co-IP of p97, mouse anti-p97 antibodies (SCBT, Cat# sc-57492) and Protein A/G magnetic S21 beads (ThermoFisher, Cat# 88802) were used to capture the p97-containing complexes. During the elution step, the beads containing p47-bound complexes were mixed at room temperature for 10 min in 1X LDS sample buffer.
For western blots, protein levels of the INPUT and UNBOUND groups were quantified using Pierce Rapid Gold BCA Protein Assay Kit (ThermoFisher, Cat# A53225) and lysates were diluted to approximately equal concentrations with NP-40 lysis buffer, followed by heating in 1X LDS sample buffer (ThermoFisher, Cat# B0007) at 90 °C for 5 min. Equal amounts of INPUT and UNBOUND samples (typically 8 μg total protein per lane) were loaded into lanes of a 12-, 15-, or 17-well Bolt 4~12% bis-tris gels (ThermoFisher, Cat# NW04122BOX, NW04125BOX, or NW04127BOX) and run at 90 V constant for 100 min. For co-IP samples, ~15 μL elution from each group was loaded in each lane. Next, protein was transferred to a PVDF membrane within the iBlot 2 mini transfer stacks (ThermoFisher, Cat # IB24002) using the iBlot 2 gel transfer device (ThermoFisher, Cat# IB21001; Condition: 20 V, 7 min). The transferred PVDF membrane was blocked with TBS blocking buffer (LI-COR Biosciences, Cat# 927-60001) for an hour at room temperature. The membrane was probed with primary antibodies that were diluted in TBS blocking buffer (contains 0.1% Tween-20) overnight at 4°C. After washing with 1X TBST (50 mM Tris·HCl, 150 mM NaCl, 0.1% Tween-20), the membrane was incubated in secondary antibodies that were diluted in TBS blocking buffer (contains 0.1% Tween-20) for 1 h at room temperature. After washing with 1X TBST, the membranes were imaged on an Odyssey CLx infrared imaging system (LI-COR Biosciences).
Cell sorting and expansion. Next, trypsinized cells were suspended in sorting buffer (PBS containing 5% FBS). A selected population of GFP-positive cells were sorted as single clones into 96-well plates (Corning, Cat# 3628) using a BD FACS Aria II sorter. The sorted single-cell clones were cultured in complete DMEM/F12 media (ThermoFisher, Cat# 10565018, supplemented with 10% FBS) for 2 weeks, with fresh DMEM/F12 media replaced on Day 5 and every 3 days afterwards.
After 2 weeks, successfully expanded clones were detached from 96-well plates and further expanded in 6-well plates until at least 80% confluency was reached.
Identification and characterization of p47-KO clones. Around 50 clones were successfully expanded to the 6-well plate stage.
These clones were subsequently harvested and analyzed by western blots. Five clones (C3, C4, C17, C34, and C45) without p47 expression were selected for genomic analysis. The genomic DNA of these clones were collected by a Quick-DNA miniprep plus kit (Zymo Research, Cat# D4069) and PCR amplified with Q5 polymerase ( Figure S11). Note that the primers for the PCR reaction were designed 500~600 bp upstream and downstream of the targeting site of p47-KO plasmid (Santa Cruz Biotechnology, Cat# sc-402328). The PCR product of these five clones along with wild-type U2OS was cleaned up (QIAGEN, Cat# 28104) and assessed by Sanger sequencing, followed by the Synthego ICE Analysis (https://ice.synthego.com/) to compare the knockout score. Clone 34 was labeled as the p47-KO U2OS cells and used for the NanoBRET assays.

Plasmid design for the NanoBRET assay
All the plasmids for the p97/p47 NanoBRET assays were constructed based on the vectors provided by the NanoBRET PPI starter system (Promega, Cat# N1811). The initial test was to screen all the possible combinations of p97/p47 pairs and find the pair that demonstrates the highest BRET signal. In this step, both p97 and p47 were tagged with a nuclear export signal (NES) SR10 at the C-terminus. The NanoLuc (NLF1) donor and HaloTag (HT) acceptor were tagged on either the N-terminus or the C-terminus of p97-NES or p47-NES, forming eight different constructs: pHTN-p97, S24 pHTC-p97, pNLF1N-p97, pNLF1C-p97, pHTN-p47, pHTC-p47, pNLF1N-p47, and pNLF1C-p47. Eight different combinations from the above eight constructs were tested for NanoBRET assays. Among the combinations (wplasmid/wplasmid = 1:1), the pair of pNLF1N-p97 (donor) and pHTC-p47 (acceptor) generated the highest BRET ratio in wild-type U2OS cells. Next, varied ratios of plasmids for pHTC-p47/pNLF1N-p97 were tested (1:1, 10:1, 100:1, and 1000:1) for NanoBRET assays, and 100:1 was selected as the ratio for the assay because of high BRET ratio and low standard deviation.
Another two pairs of p97/p47 constructs were also generated: (1) Full-length p97 and p47 without NES tag; (2) p97-N domain with NLF1 tagged on the N-terminus (pNLF1N-p97-N) and p47-UBX domain with HT tagged on the C-terminus (pHTC-p47-UBX). These two pairs generated similar BRET signals when compared to the full-length constructs with the NES tag ( Figure S12).

General procedures of the NanoBRET assay
A total of 120 k U2OS cells (wild-type or p47-KO) in complete DMEM media (w/o antibiotics) were cultured in a 6-well plate (Corning,Cat# 3516) for 24 hours prior to the experiment. The complexes between 1.5 μL Xfect transfection reagent (Takara Bio, Cat# 631318) and 5 μg plasmids [5 μg NanoBRET pair plasmids in total, or 2.5 μg NanoBRET pair plasmids along with 2.5 μg plasmids for antibodies/empty vector (EV; ThermoFisher, Cat# V79020)/p47-FLAG] were mixed in 100 μL Xfect reaction buffer for 10 min. After replacing the media with 900 μL fresh complete DMEM media (w/o antibiotics), the Xfect-plasmid mixture was added dropwise into the well and incubated at 37 °C. After 24 hours, each group of the transfected cells were detached and resuspended in FluoroBrite DMEM (ThermoFisher, Cat# A1896701; supplemented with 10% FBS, 1X antibiotics, and 1X GlutaMAX). Next, ~24 k transfected cells in 100 μL FluoroBrite DMEM were seeded in 96-well plates (Corning,Cat# 3610). In each well, the experimental samples were added with 0.1 μL stock solution of the HaloTag NanoBRET 618 Ligand (the "+ligand" group), and the no-acceptor controls were added with 0.1 μL DMSO (the "-ligand" group). After incubating at 37 °C for 6 hours, 25 μL 5X solution NanoBRET Nano-Glo Substrate reagent in FluoroBrite DMEM was spiked into each well and immediately measured the luminescence on a